1. Field of the Invention
The invention relates to nucleic acid based kits and methods for determining the presence or absence in a sample of respiratory pathogens including the following: influenza A (including subtyping capability for H1, H3, H5 and H7 subtypes) influenza B, parainfluenza (type 2), respiratory syncytial virus, and adenovirus.
2. Description of the Related Art
During flu season, as many as half of adult patients admitted to the emergency room are admitted with respiratory complaints. Accurate diagnosis of the patient requires analysis of clinical samples. Clinical samples are generally obtained as nasopharyngeal or throat swabs, nasal aspirate, or nasal washes, and are analyzed using viral culture, enzyme immunoassay (EIA), direct immunofluorescence antibody staining (DFA), or reverse transcriptase-polymerase chain reaction (RT-PCR).
Viral culture (the gold standard) is both sensitive and specific, but it requires 3-10 days to provide results, far too late to establish the cause of an outbreak of respiratory illness for early intervention; the method is also labor-intensive. EIA and optical immunoassay can provide rapid results (30 minutes), but the assays lack adequate sensitivity and specificity. DFA exhibits sensitivity comparable to viral culture.
DFA reagents are the mainstay of respiratory virus detection in many hospitals since reagents can detect more than one respiratory pathogen simultaneously (i.e., multiplexed) from a single sample. Multiplexed assays have been developed for detection of the most common respiratory diseases, including influenza A and B, respiratory syncytial virus (RSV), parainfluenza (Types 1-3) and adenovirus. Results can be obtained in 1-2 hours. DFA, however, requires samples with adequate numbers of target cells, high-quality equipment, a skilled microscopist, and is ultimately labor-intensive and subjective, making it less suitable for use in reference laboratories.
Many groups have demonstrated that the sensitivity and specificity of RT-PCR assays for Influenza A and B are on par with viral culture and DFA; results can be obtained in 2 hours, and large numbers of samples can be rapidly tested; however, multiplexed RT-PCR assays are not available.
A number of rapid diagnostic test kits for detection of influenza are commercially available (e.g., Becton-Dickenson Directagen Flu A, B-D Directagen Flu A+B, Binax NOW Flu Test, ZymeTx ZstatFlu). The rapid test kits generally provide results within 24 hours and are approximately 70% sensitive for detecting influenza and approximately 90% specific. The sensitivity of the rapid test kits means that as many as 30% of samples may yield false negatives, and the tests are not multiplexed.
Each of these assay techniques described above has disadvantages that make them more or less suitable for use in public health laboratories, or hospital-based laboratories, but none of these existing assays are currently employed at point-of care. They all conducted in a laboratory and usually results are not produced rapidly enough to impact on the prescribed treatment.
Accordingly, there exists a significant need for rapid and accurate multiplex tests for identification of respiratory pathogens.
Traditional approaches to DNA signature development started with the hypothesis that a particular gene was vital to the organism's virulence, host range, or other factors that might be considered “unique”. Suitable primers and probe were designed for the detection system of choice, with or without computational screening (via BLAST or equivalent) for uniqueness. The resulting assay would then be tested with the available strain(s) and success declared if the targets were detected, but the assay didn't detect whatever near-neighbors were tested. This approach would sometimes yield good results, but failures occurred due to inadequate strain panel coverage and cross reactions with genetic near neighbors and complex environmental samples.